Advances in Vaccines against Infectious Diseases

A special issue of Vaccines (ISSN 2076-393X). This special issue belongs to the section "Vaccines against Tropical and other Infectious Diseases".

Deadline for manuscript submissions: 31 December 2024 | Viewed by 11255

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Guest Editor
Center for Technological Development in Health (CDTS), National Institute of Science and Technology for Innovation in Neglected Diseases Populations (INCT-IDPN), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro 21040-900, RJ, Brazil
Interests: vaccines; infectious diseases; molecular immunology; epitopes; neglected diseases; biochemistry; molecular biology; proteins; structure; cell biology
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Special Issue Information

Dear Colleagues,

The occurrence of infectious diseases still has a significant impact on public health and the economy. Infectious diseases spread from person to person, and it is important to control the spread of these diseases globally to ensure the well-being of everyone and a country's economic prosperity. Vaccination is an important step towards achieving universal health coverage. Recent outbreaks of emerging, re-emerging, and neglected infectious diseases show the need for effective control and prevention measures, including advancements in vaccine development, adjuvants, and immunity studies to understand the protective effects of the vaccines better. This Special Issue invites researchers to contribute their insights and on vaccines and vaccination, exploring vaccines and emerging, re-emerging, and neglected infectious diseases. Original articles, perspectives, and reviews on vaccine studies, viral immunology, and vaccine tools that facilitate examining these interactions at an immunological molecular level are welcome.

Dr. Salvatore Giovanni De Simone
Guest Editor

Manuscript Submission Information

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Keywords

  • vaccination
  • antigens
  • disease
  • immune
  • immunity
  • vaccine delivery
  • host responses
  • biodefence
  • emerging pathogens
  • MERS
  • parasites
  • virus
  • bacteria
  • adjuvants

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Published Papers (10 papers)

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Research

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8 pages, 508 KiB  
Communication
Differential Neutralization Profiles of 17DD Vaccinated Population to 17D-204 and 17DD Vaccine Strains
by Ana C. B. Terzian, Sasha R. Azar, Cassia F. Estofolete, Mauricio L. Nogueira and Nikos Vasilakis
Vaccines 2024, 12(12), 1311; https://doi.org/10.3390/vaccines12121311 (registering DOI) - 23 Nov 2024
Abstract
Background/Objectives: Yellow fever virus (YFV) (Flaviviridae, Orthoflavivirus) is the etiologic agent of yellow fever (YF), a vector-borne disease with significant morbidity and mortality across the tropics and neotropics, despite having a highly efficacious and safe vaccine (17D). Vaccination provides [...] Read more.
Background/Objectives: Yellow fever virus (YFV) (Flaviviridae, Orthoflavivirus) is the etiologic agent of yellow fever (YF), a vector-borne disease with significant morbidity and mortality across the tropics and neotropics, despite having a highly efficacious and safe vaccine (17D). Vaccination provides lifelong protection from YF disease mediated by humoral immunity. There are several versions of the original 17D vaccine: 17D-204 (marketed in the USA as YF-VAX, in France as Stamaril, and in China as Tiantan-V), 17D-213 (Russian Federation), and 17DD (by FIOCRUZ in Brazil). Vaccines produced in the US, France, Senegal, China, and Russia represent 17D-204-derived strains, whereas the Brazilian 17DD has a unique passage/attenuation history from 17D-204-derived strains. Their functional differences in the neutralization profiles are not known. Methods: The Plaque Reduction Neutralization Test (PRNT) was used to determine the neutralization profiles of sera from 209 patients that were previously vaccinated with the 17DD strain against both 17D-204 and 17DD. Results: Sera exhibited significantly more efficient neutralization of 17DD (mean reciprocal PRNT50 183, PRNT80 86, median reciprocal PRNT50 80, and PRNT80 40) compared to 17D-204 (mean reciprocal PRNT50 91, PRNT80 33, median reciprocal PRNT50 40, and PRNT80 10). Conclusions: Our data indicate antigenic differences between 17D and 17DD vaccines. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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15 pages, 442 KiB  
Article
The Influence of Health Education on Vaccination Coverage and Knowledge of the School Population Related to Vaccination and Infection Caused by the Human Papillomavirus
by Ivana Kotromanović Šimić, Vesna Bilić-Kirin, Maja Miskulin, Darko Kotromanović, Marija Olujić, Jelena Kovacevic, Danijela Nujić, Nika Pavlovic, Ivan Vukoja and Ivan Miskulin
Vaccines 2024, 12(11), 1222; https://doi.org/10.3390/vaccines12111222 - 28 Oct 2024
Viewed by 624
Abstract
Background/Objectives: Human papillomavirus (HPV) is a causative agent of infections and cancers of the reproductive and digestive tract, and vaccination is the most effective prevention method. This research aimed to assess the impact of health education on vaccination coverage and knowledge of the [...] Read more.
Background/Objectives: Human papillomavirus (HPV) is a causative agent of infections and cancers of the reproductive and digestive tract, and vaccination is the most effective prevention method. This research aimed to assess the impact of health education on vaccination coverage and knowledge of the school population related to HPV infection and vaccination. Methods: This non-randomized clinical trial included 170 participants out of the 221 students in the generation of 2022/2023, who were divided into a group of Subjects and Controls and who self-assessed their knowledge and tested knowledge at four and two time points, respectively. Results: The study examined whether there is a difference in the share of vaccinated students of the entire generation compared to the previous generation (62% vs. 47%). The self-assessment and assessment of one’s knowledge in the group of subjects was significantly lower before education than during other time points during the research. At “moment zero”, there was no significant difference in the knowledge assessment between the groups. However, after 8 weeks, the knowledge assessment was significantly higher in the Subject group. Conclusions: The results suggest a positive impact of health education, which may contribute to raising awareness about the importance of prevention and vaccination against HPV. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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23 pages, 5229 KiB  
Article
Immunoreactivity Analysis of MHC-I Epitopes Derived from the Nucleocapsid Protein of SARS-CoV-2 via Computation and Vaccination
by Dongbo Jiang, Zilu Ma, Junqi Zhang, Yubo Sun, Tianyuan Bai, Ruibo Liu, Yongkai Wang, Liang Guan, Shuaishuai Fu, Yuanjie Sun, Yuanzhe Li, Bingquan Zhou, Yulin Yang, Shuya Yang, Yuanhang Chang, Baozeng Sun and Kun Yang
Vaccines 2024, 12(11), 1214; https://doi.org/10.3390/vaccines12111214 - 25 Oct 2024
Viewed by 436
Abstract
Background: Since 2019, the SARS-CoV-2 virus has been responsible for the global spread of respiratory illness. As of 1 September 2024, the cumulative number of infections worldwide exceeded 776 million. There are many structural proteins of the virus, among which the SARS-CoV-2 [...] Read more.
Background: Since 2019, the SARS-CoV-2 virus has been responsible for the global spread of respiratory illness. As of 1 September 2024, the cumulative number of infections worldwide exceeded 776 million. There are many structural proteins of the virus, among which the SARS-CoV-2 nucleocapsid (N) protein plays a pivotal role in the viral life cycle, participating in a multitude of essential activities following viral invasion. An important antiviral immune response is the major histocompatibility complex (MHC)-restricted differentiation cluster 8 (CD8+) T cell cytotoxicity. Therefore, understanding the immunogenicity of SARS-CoV-2 NP-specific MHC-I-restricted epitopes is highly important. Methods: MHC-I molecules from 11 human leukocyte antigen I (HLA-I) superfamilies with 98% population coverage and 6 mouse H2 alleles were selected. The affinity were screened by IEDB, NetMHCpan, SYFPEITHI, SMMPMBEC and Rankpep. Further immunogenicity and conservative analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis was performed. Selective epitopes were validated by enzyme-linked immunospot (ELISpot) assay and flow cytometry in the mice with pVAX-NPSARS-CoV-2 immunization. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect whether the preferred epitope induced humoral immunity. Results: There were 64 dominant epitopes for the H-2 haplotype and 238 dominant epitopes for the HLA-I haplotype. Further analysis of immunogenicity and conservation yielded 8 preferred epitopes, and docking simulations were conducted with corresponding MHC-I alleles. The relationships between the NP peptides and MHC-I haplotypes were then determined via two-way hierarchical clustering. ELISA, ELISpot assay, and flow cytometry revealed that the preferred epitope stimulated both humoral and cellular immunity and enhanced cytokine secretion in mice. Conclusions: our study revealed the general patterns among multiple haplotypes within the humans and mice superfamily, providing a comprehensive assessment of the pan-MHC-I immunoreactivity of SARS-CoV-2 NP. Our findings would render prospects for the development and application of epitope-based immunotherapy in lasting viral epidemics. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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12 pages, 1475 KiB  
Article
Humoral Immunity and Antibody Responses against Diphtheria, Tetanus, and Pneumococcus after Immune Effector Cell Therapies: A Prospective Study
by Georgios Angelidakis, Roy F. Chemaly, Pranoti V. Sahasrabhojane, Oscar Morado-Aramburo, Ying Jiang, Micah M. Bhatti, Elizabeth Shpall, Chitra Hosing, Preetesh Jain, Kris Michael Mahadeo, Fareed Khawaja, Peter Elhajj, Jennifer A. Wargo, Robert R. Jenq, Nadim J. Ajami, Partow Kebriaei and Ella J. Ariza-Heredia
Vaccines 2024, 12(9), 1070; https://doi.org/10.3390/vaccines12091070 - 19 Sep 2024
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Abstract
Patients undergoing immune effector cell therapy (IECT) are at high risk for infections. We assessed seropositivity against pneumococcus, tetanus, and diphtheria in patients before and after IECT and the patients’ response to vaccination. We enrolled patients who underwent IECT from January 2020 to [...] Read more.
Patients undergoing immune effector cell therapy (IECT) are at high risk for infections. We assessed seropositivity against pneumococcus, tetanus, and diphtheria in patients before and after IECT and the patients’ response to vaccination. We enrolled patients who underwent IECT from January 2020 to March 2022. Antibody levels for diphtheria, tetanus, and pneumococcus were measured before IECT, at 1 month, and 3–6 months after. Eligible patients were vaccinated after IECT. In non-seroprotected patients, we discontinued testing. Before IECT, most patients had seroprotective antibody levels against tetanus (68/69, 99%) and diphtheria (65/69, 94%), but fewer did against pneumococcus (24/67, 36%). After IECT, all patients had seroprotective antibody levels for tetanus at 1 month (68/68) and 3–6 months (56/56). For diphtheria, 65/65 patients (100%) had seroprotective antibody levels at 1 month, and 48/53 (91%) did at 3–6 months. For pneumococcus, seroprotective antibody levels were identified in 91% (21/23) of patients at 1 month and 79% (15/19) at 3–6 months following IECT. Fifteen patients received a pneumococcal vaccine after IECT, but none achieved seroprotective response. One patient received the tetanus-diphtheria vaccine and had a seroprotective antibody response. Because some patients experience loss of immunity after IECT, studies evaluating vaccination strategies post-IECT are needed. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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17 pages, 2725 KiB  
Article
Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP
by Andressa da M. Durans, Paloma Napoleão-Pêgo, Flavia C. G. Reis, Evandro R. Dias, Luciana E. S. F. Machado, Guilherme C. Lechuga, Angela C. V. Junqueira, Salvatore G. De-Simone and David W. Provance, Jr.
Vaccines 2024, 12(9), 1029; https://doi.org/10.3390/vaccines12091029 - 8 Sep 2024
Cited by 1 | Viewed by 1050
Abstract
Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have [...] Read more.
Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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14 pages, 19599 KiB  
Article
Lysosome-Associated Membrane Protein Targeting Strategy Improved Immunogenicity of Glycoprotein-Based DNA Vaccine for Marburg Virus
by Xiyang Zhang, Yubo Sun, Junqi Zhang, Hengzheng Wei, Jing Wang, Chenchen Hu, Yang Liu, Sirui Cai, Qinghong Yuan, Yueyue Wang, Yuanjie Sun, Shuya Yang, Dongbo Jiang and Kun Yang
Vaccines 2024, 12(9), 1013; https://doi.org/10.3390/vaccines12091013 - 4 Sep 2024
Viewed by 893
Abstract
Marburg hemorrhagic fever (MHF) is a fatal infectious disease caused by Marburg virus (MARV) infection, and MARV has been identified as a priority pathogen for vaccine development by the WHO. The glycoprotein (GP) of MARV mediates viral adhesion and invasion of host cells [...] Read more.
Marburg hemorrhagic fever (MHF) is a fatal infectious disease caused by Marburg virus (MARV) infection, and MARV has been identified as a priority pathogen for vaccine development by the WHO. The glycoprotein (GP) of MARV mediates viral adhesion and invasion of host cells and therefore can be used as an effective target for vaccine development. Moreover, DNA vaccines have unique advantages, such as simple construction processes, low production costs, and few adverse reactions, but their immunogenicity may decrease due to the poor absorption rate of plasmids. Lysosome-associated membrane protein 1 (LAMP1) can direct antigens to lysosomes and endosomes and has great potential for improving the immunogenicity of nucleic acid vaccines. Therefore, we constructed a DNA vaccine based on a codon-optimized MARV GP (ID MF939097.1) fused with LAMP1 and explored the effect of a LAMP targeting strategy on improving the immunogenicity of the MARV DNA vaccine. ELISA, ELISpot, and flow cytometry revealed that the introduction of LAMP1 into the MARV DNA candidate vaccine improved the humoral and cellular immune response, enhanced the secretion of cytokines, and established long-term immune protection. Transcriptome analysis revealed that the LAMP targeting strategy significantly enriched antigen processing and presentation-related pathways, especially the MHC class II-related pathway, in the candidate vaccine. Our study broadens the strategic vision for enhanced DNA vaccine design and provides a promising candidate vaccine for MHF prevention. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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15 pages, 698 KiB  
Article
Safety and Immunogenicity of the Nonavalent Human Papillomavirus Vaccine in Women Living with HIV
by Carmen Hidalgo-Tenorio, Raquel Moya, Mohamed Omar, Leopoldo Muñoz, Antonio SamPedro, Javier López-Hidalgo, Coral Garcia-Vallecillos and Patricia Gómez-Ronquillo
Vaccines 2024, 12(8), 838; https://doi.org/10.3390/vaccines12080838 - 25 Jul 2024
Cited by 1 | Viewed by 1180
Abstract
Background: The objectives were to evaluate the safety and immunogenicity of the nonavalent human papillomavirus (nHPV) vaccine in adult Spanish women living with HIV (WLHIV); the prevalence of anal and cervical dysplasia and nHPV vaccine genotypes in the anus and cervix; and risk [...] Read more.
Background: The objectives were to evaluate the safety and immunogenicity of the nonavalent human papillomavirus (nHPV) vaccine in adult Spanish women living with HIV (WLHIV); the prevalence of anal and cervical dysplasia and nHPV vaccine genotypes in the anus and cervix; and risk factors for high-risk HPV (HR-HPV) infection in anal mucosa. Methods: In this single-center, open-arm, non-randomized clinical trial, the nHPV vaccine was administered at 0, 2, and 6 months to WLHIV enrolled between February 2020 and November 2023, measuring vaccine antibody titers pre-vaccination and at 2, 6, and 7 months after the first dose. Cervical and anal cytology and HPV PCR genotyping studies were performed. Women with abnormal cytology and/or anal or cervical HPV infection at baseline underwent high-resolution anoscopy and/or colposcopy. Results: A total of 122 participants were included with mean age of 49.6 years: 52.5% smoked; 10.7% had anal-genital condylomatosis; 38.5% were infected by HR-HPV in the anus and 25.4% in the cervix, most frequently HPV 16; 19.1% had anal intraepithelial neoplasia 1-(AIN1); and 3.1% had cervical intraepithelial neoplasia 1 and 2 (CIN1/CIN2). Vaccine administration did not modify viral–immunological status (CD4 [809 ± 226.8 cells/uL vs. 792.35 ± 349.95; p = 0.357]) or plasma HIV load (3.38 ± 4.41 vs. 1.62 ± 2.55 cop/uL [log]; p = 0.125). Anti-HPV antibodies ([IQR: 0–0] vs. 7.63 nm [IQR: 3.46–19.7]; p = 0.0001) and seroconversion rate (8.2% vs. 96.7% [p = 0.0001]) were increased at 7 versus 0 months. There were no severe vaccine-related adverse reactions; injection-site pain was reported by around half of the participants. HR-HPV infection in the anus was solely associated with a concomitant cervix infection (HR 5.027; 95% CI: 1.009–25.042). Conclusions: nHPV vaccine in adult WLHIV is immunogenic and safe. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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16 pages, 5701 KiB  
Article
Production of Promising Heat-Labile Enterotoxin (LT) B Subunit-Based Self-Assembled Bioconjugate Nanovaccines against Infectious Diseases
by Caixia Li, Juntao Li, Peng Sun, Ting Li, Xue Yan, Jingqin Ye, Jun Wu, Li Zhu, Hengliang Wang and Chao Pan
Vaccines 2024, 12(4), 347; https://doi.org/10.3390/vaccines12040347 - 23 Mar 2024
Viewed by 1481
Abstract
Nanoparticles (NPs) have been widely utilized in vaccine design. Although numerous NPs have been explored, NPs with adjuvant effects on their own have rarely been reported. We produce a promising self-assembled NP by integrating the pentameric Escherichia coli heat-labile enterotoxin B subunit (LTB) [...] Read more.
Nanoparticles (NPs) have been widely utilized in vaccine design. Although numerous NPs have been explored, NPs with adjuvant effects on their own have rarely been reported. We produce a promising self-assembled NP by integrating the pentameric Escherichia coli heat-labile enterotoxin B subunit (LTB) (studied as a vaccine adjuvant) with a trimer-forming peptide. This fusion protein can self-assemble into the NP during expression, and polysaccharide antigens (OPS) are then loaded in vivo using glycosylation. We initially produced two Salmonella paratyphi A conjugate nanovaccines using two LTB subfamilies (LTIB and LTIIbB). After confirming their biosafety in mice, the data showed that both nanovaccines (NP(LTIB)-OPSSPA and NP(LTIIbB)-OPSSPA) elicited strong polysaccharide-specific antibody responses, and NP(LTIB)-OPS resulted in better protection. Furthermore, polysaccharides derived from Shigella or Klebsiella pneumoniae were loaded onto NP(LTIB) and NP(LTIIbB). The animal experimental results indicated that LTIB, as a pentamer module, exhibited excellent protection against lethal infections. This effect was also consistent with that of the reported cholera toxin B subunit (CTB) modular NP in all three models. For the first time, we prepared a novel promising self-assembled NP based on LTIB. In summary, these results indicated that the LTB-based nanocarriers have the potential for broad applications, further expanding the library of self-assembled nanocarriers. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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12 pages, 6196 KiB  
Article
Humoral Immune Response to SARS-CoV-2 Spike Protein Receptor-Binding Motif Linear Epitopes
by Maria E. S. Monteiro, Guilherme C. Lechuga, Paloma Napoleão-Pêgo, João P. R. S. Carvalho, Larissa R. Gomes, Carlos M. Morel, David W. Provance and Salvatore G. De-Simone
Vaccines 2024, 12(4), 342; https://doi.org/10.3390/vaccines12040342 - 22 Mar 2024
Cited by 2 | Viewed by 1784
Abstract
The worldwide spread of SARS-CoV-2 has led to a significant economic and social burden on a global scale. Even though the pandemic has concluded, apprehension remains regarding the emergence of highly transmissible variants capable of evading immunity induced by either vaccination or prior [...] Read more.
The worldwide spread of SARS-CoV-2 has led to a significant economic and social burden on a global scale. Even though the pandemic has concluded, apprehension remains regarding the emergence of highly transmissible variants capable of evading immunity induced by either vaccination or prior infection. The success of viral penetration is due to the specific amino acid residues of the receptor-binding motif (RBM) involved in viral attachment. This region interacts with the cellular receptor ACE2, triggering a neutralizing antibody (nAb) response. In this study, we evaluated serum immunogenicity from individuals who received either a single dose or a combination of different vaccines against the original SARS-CoV-2 strain and a mutated linear RBM. Despite a modest antibody response to wild-type SARS-CoV-2 RBM, the Omicron variants exhibit four mutations in the RBM (S477N, T478K, E484A, and F486V) that result in even lower antibody titers. The primary immune responses observed were directed toward IgA and IgG. While nAbs typically target the RBD, our investigation has unveiled reduced seroreactivity within the RBD’s crucial subregion, the RBM. This deficiency may have implications for the generation of protective nAbs. An evaluation of S1WT and S2WT RBM peptides binding to nAbs using microscale thermophoresis revealed a higher affinity (35 nM) for the S2WT sequence (GSTPCNGVEGFNCYF), which includes the FNCY patch. Our findings suggest that the linear RBM of SARS-CoV-2 is not an immunodominant region in vaccinated individuals. Comprehending the intricate dynamics of the humoral response, its interplay with viral evolution, and host genetics is crucial for formulating effective vaccination strategies, targeting not only SARS-CoV-2 but also anticipating potential future coronaviruses. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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Review

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12 pages, 986 KiB  
Review
Mycobacterium tuberculosis–Human Immunodeficiency Virus Infection and the Role of T Cells in Protection
by Kiana Hosseinian, Amir Gerami, Melody Bral and Vishwanath Venketaraman
Vaccines 2024, 12(7), 730; https://doi.org/10.3390/vaccines12070730 - 30 Jun 2024
Viewed by 1692
Abstract
Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (M. tb), remains a widespread fatal health issue that becomes significantly detrimental when coupled with HIV. This study explores the host’s innate and adaptive immune system response to TB in HIV immunocompromised patients, highlighting [...] Read more.
Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (M. tb), remains a widespread fatal health issue that becomes significantly detrimental when coupled with HIV. This study explores the host’s innate and adaptive immune system response to TB in HIV immunocompromised patients, highlighting the significant role of CD8+ T cells. While the crucial role of macrophages and cytokines, like TNF-α and IFN-γ, in managing the host’s immune response to M. tb is examined, the emphasis is on the changes that occur as a result of HIV coinfection. With the progression of HIV infection, the primary source of IFN-γ changes from CD4+ to CD8+ T cells, especially when latent TB advances to an active state. This study sheds light on the necessity of developing new preventative measures such as vaccines and new treatment approaches to TB, especially for immunocompromised patients, who are at a higher risk of life-threatening complications due to TB-HIV coinfection. Full article
(This article belongs to the Special Issue Advances in Vaccines against Infectious Diseases)
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